Microfluidic Formation of Ionically Cross-Linked Polyamine Gels

Jan 15, 2010 - Stefano Menegatti , Nino Ruocco , Sunny Kumar , Michael Zakrewsky , Joshua Sanchez ... Serdar Ozturk , Yassin A. Hassan , Victor M. Uga...
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Microfluidic Formation of Ionically Cross-Linked Polyamine Gels Gautam C. Kini,† Justin Lai,‡ Michael S. Wong,*,†,§ and Sibani Lisa Biswal*,† †

Department of Chemical and Biomolecular Engineering, ‡Department of Biochemistry and Cell Biology, and § Department of Chemistry, Rice University, Houston, Texas Received October 20, 2009. Revised Manuscript Received December 17, 2009

In this article, we discuss in situ polymer gelation in microfluidic channels from electrostatically mediated interactions when reactant streams of a linear cationic polymer (poly(allylamine hydrochloride, PAH) and a multivalent anion (sodium citrate) are subjected to shear flow. We find that the polyamine exhibits shear-thickening behavior as it is ionically crosslinked by citrate ions to form viscoelastic gel phases. These gels form at room temperature and remain stable and intact after the cessation of flow. Gelation is found to occur in the polymer stream and not the citrate stream because of an appreciably higher diffusivity of citrate ions when compared to the gel and PAH and because of laminar flow conditions in the microfluidic environment. Gel formation occurred when the pH of the PAH stream was below the PAH pKa value of 8.38 and when citrate was either in a disodium or trisodium state. The formation of aggregates, gels, and droplets was found to depend strongly on the charge ratio and flow conditions. The gelation of PAH begins with the formation of colloidal aggregates of PAH and citrate, which then combine under shear flow to form noncontinuous or continuous gels. Droplets of citrate can form within regions of continuous gels as excess citrate anions diffuse into the gel stream.

1. Introduction A recent focus area in microfluidic research has been on flow and the formation of gels in microchannels.1-8 Microfluidic devices allow for the confinement of reagents and precise *Corresponding authors. E-mail: [email protected]; [email protected]. (1) Roberts, M. T.; Mohraz, A.; Christensen, K. T.; Lewis, J. A. Langmuir 2007, 23, 8726–8731. (2) Conrad, J. C.; Lewis, J. A. Langmuir 2008, 24, 7628–7634. (3) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B. H. Nature 2000, 404, 588–590. (4) Amici, E.; Tetradis-Meris, G.; de Torres, P.; Jousse, F. Food Hydrocolloids 2008, 22, 97–104. (5) Koh, W. G.; Pishko, M. Langmuir 2003, 19, 10310–10316. (6) Bazargan, V.; Stoeber, B. Phys. Rev. E 2008, 78, 066303. (7) De Geest, B. G.; Urbanski, J. P.; Thorsen, T.; Demeester, J.; De Smedt, S. C. Langmuir 2005, 21, 10275–10279. (8) Steinbacher, J. L.; McQuade, D. T. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 6505–6533. (9) Hernandez-Ortiz, J. P.; Ma, H. B.; Pablo, J. J.; Graham, M. D. Phys. Fluids 2006, 18, 123101. (10) deMello, A. J. Nature 2006, 442, 394–402. (11) Shestopalov, I.; Tice, J. D.; Ismagilov, R. F. Lab Chip 2004, 4, 316–321. (12) Caulum, M. M.; Henry, C. S. Lab Chip 2008, 8, 865–867. (13) Ahmed-Omer, B.; Brandt, J. C.; Wirth, T. Org. Biomol. Chem. 2007, 5, 733–740. (14) Ristenpart, W. D.; Wan, J. D.; Stone, H. A. Anal. Chem. 2008, 80, 3270– 3276. (15) Serra, C. A.; Chang, Z. Q. Chem. Eng. Technol. 2008, 31, 1099–1115. (16) Zhang, H.; Tumarkin, E.; Sullan, R. M. A.; Walker, G. C.; Kumacheva, E. Macromol. Rapid Commun. 2007, 28, 527–538. (17) Calvert, P. MRS Bull. 2008, 33, 207–212. (18) Zhang, C. S.; Xing, D.; Li, Y. Y. Biotechnol. Adv. 2007, 25, 483–514. (19) Zhang, Y.; Kato, S.; Anazawa, T. Sens. Actuators, B 2008, 129, 481–486. (20) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Frechett, J. M. J. Anal. Chem. 2003, 75, 1958–1961. (21) Harmon, M. E.; Tang, M.; Frank, C. W. Polymer 2003, 44, 4547–4556. (22) Luo, Q. Z.; Mutlu, S.; Gianchandani, Y. B.; Svec, F.; Frechet, J. M. J. Electrophoresis 2003, 24, 3694–3702. (23) Sugiura, S.; Sumaru, K.; Ohi, K.; Hiroki, K.; Takagi, T.; Kanamori, T. Sens. Actuators, A 2007, 140, 176–184. (24) Siegel, R. A.; Gu, Y. D.; Baldi, A.; Ziaie, B. Macromol. Symp. 2004, 207, 249–256. (25) De, S. K.; Aluru, N. R.; Johnson, B.; Crone, W. C.; Beebe, D. J. J. Microelectromech. Syst. 2002, 11, 544–555. (26) De, S. K.; Aluru, N. R. Mech. Mater. 2004, 36, 395–410. (27) Osada, Y.; Gong, J. P. Prog. Polym. Sci. 1993, 18, 187–226. (28) Good, B. T.; Bowman, C. N.; Davis, R. H. Chem. Eng. Sci. 2004, 59, 5967– 5974.

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control of the time of reaction.9-16 Primary applications of gels in microfluidic devices are in forming components for flow control,3,17-30 separations,31-35 detection, and sensing,24,33 which are essential in developing micrototal analytical (μTAS) systems. Recent μTAS systems that use gel-based components include glucose and insulin detection devices,19,24 enzymatic microreactors,36 optical chemosensors,37 and nucleic acid separation systems.38 An effective approach to incorporating gels within a microchannel is to form gel structures in situ.3,29,30,39 In situ gel formation is an important route to fabricating gels at precise locations in a microchannel, which otherwise would require the use of multiple microfabrication steps.3,5 To date, in situ gel formation in microfluidic channels have been based on polymerization reactions, in combination with additional steps such as flow focusing, geometric confinement, and UV/photolithography.3,7,29,30,40-54 In some cases, charge-based cross-linking (29) Kim, D.; Beebe, D. J. Sens. Actuators, A 2007, 136, 426–433. (30) Kim, D.; Beebe, D. J. Lab Chip 2007, 7, 193–198. (31) Slater, G. W.; Guillouzic, S.; Gauthier, M. G.; Mercier, J. F.; Kenward, M.; McCormick, L. C.; Tessier, F. Electrophoresis 2002, 23, 3791–3816. (32) Li, Y.; Buch, J. S.; Rosenberger, F.; DeVoe, D. L.; Lee, C. S. Anal. Chem. 2004, 76, 742–748. (33) Vegvari, A.; Hjerten, S. Electrophoresis 2002, 23, 3479–3486. (34) Viovy, J. L. Rev. Mod. Phys. 2000, 72, 813–872. (35) Chen, G.; Xu, X. J.; Lin, Y. H.; Wang, J. Chem.;Eur. J. 2007, 13, 6461– 6467. (36) Qu, H. Y.; Wang, H. T.; Huang, Y.; Zhong, W.; Lu, H. J.; Kong, J. L.; Yang, P. Y.; Liu, B. H. Anal. Chem. 2004, 76, 6426–6433. (37) Rudzinski, C. M.; Young, A. M.; Nocera, D. G. J. Am. Chem. Soc. 2002, 124, 1723–1727. (38) Chan, A.; Krull, U. J. Anal. Chim. Acta 2006, 578, 31–42. (39) Peterson, D. S. Lab Chip 2005, 5, 132–139. (40) Lewis, P. C.; Graham, R. R.; Nie, Z. H.; Xu, S. Q.; Seo, M.; Kumacheva, E. Macromolecules 2005, 38, 4536–4538. (41) Bouquey, M.; Serra, C.; Berton, N.; Prat, L.; Hadziioannou, G. Chem. Eng. J. 2008, 135, S93–S98. (42) Randall, G. C.; Schultz, K. M.; Doyle, P. S. Lab Chip 2006, 6, 516–525. (43) Zourob, M.; Mohr, S.; Mayes, A. G.; Macaskill, A.; Perez-Moral, N.; Fielden, P. R.; Goddard, N. J. Lab Chip 2006, 6, 296–301. (44) Nisisako, T.; Torii, T. Adv. Mater. 2007, 19, 1489–1493. (45) Rondeau, E.; Cooper-White, J. J. Langmuir 2008, 24, 6937–6945. (46) Nie, Z. H.; Xu, S. Q.; Seo, M.; Lewis, P. C.; Kumacheva, E. J. Am. Chem. Soc. 2005, 127, 8058–8063.

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reactions have also been used to create gels and gel-based structures, such as those prepared from calcium chloride and sodium alginate.4,16,55 However, even electrostatic cross-linking routes to gel formation require it to be carried out in flow-focusing geometry or with an additional polymerization step.4,45,49,55-57 Bazargan and Stoeber recently reported on reversible gelation in microchannels from thermoresponsive pluronics with salt solutions of Na3PO4. Gelation at room temperature was achieved, although it was found to occur within a limited range of pluronic and salt concentrations.6,58 In this study, we demonstrate that in situ polymer gelation can be achieved in a microfluidic channel and at room temperature using streams of cationic poly(allylamine hydrochloride) (PAH) and citrate anions. This is the first example of in situ, in-channel, room-temperature polymer gel formation that is based on electrostatic interactions and does not require an immiscible phase or a flow-focusing device. Previous investigations of interactions between PAH with multivalent anions were carried out in batch mode and under vortex-mixing conditions. It was established that the cross-linking of the polymer occurred by salt-induced counterion condensation of the polymer backbone, resulting in the formation of aggregates.59-63 This study illustrates that under continuous flow conditions and under the confinement of laminar flow, structures such as gels and droplets can be formed in addition to previously reported aggregates. Formation parameters such as the charge ratio, flow shear stresses, and pH, which influences the valency of the multivalent anion, are seen to influence gel morphology. We find that the polyamine exhibits shear-thickening behavior while cross-linking with citrate to form viscoelastic gel phases and that the gels remain stable and intact after the cessation of flow.

2. Experimental Section 2.1. Materials. Chemicals used in microfluidic template fabrication, channel fabrication, and PAH-citric acid interaction studies were procured from chemical suppliers and used without any further purification. Hydrogen peroxide (H2O2, 30% (v/v)) and sulfuric acid (H2SO4) were procured from Fisher Scientific. A piranha solution used to clean silicon wafers was prepared by mixing H2O2 and H2SO4 in a volume ratio of 1:1. The piranha (47) Seo, M.; Nie, Z. H.; Xu, S. Q.; Lewis, P. C.; Kumacheva, E. Langmuir 2005, 21, 4773–4775. (48) Nie, Z. H.; Li, W.; Seo, M.; Xu, S.; Kumacheva, E. J. Am. Chem. Soc. 2006, 128, 9408–9412. (49) Sauter, C.; Dhouib, K.; Lorber, B. Cryst. Growth Des. 2007, 7, 2247–2250. (50) Oh, H. J.; Kim, S. H.; Baek, J. Y.; Seong, G. H.; Lee, S. H. J. Micromech. Microeng. 2006, 16, 285–291. (51) Chang, J. Y.; Yang, C. H.; Huang, K. S. Nanotechnology 2007, 18, 305305. (52) Shah, R. K.; Kim, J. W.; Agresti, J. J.; Weitz, D. A.; Chu, L. Y. Soft Matter 2008, 4, 2303–2309. (53) Kim, J. W.; Utada, A. S.; Fernandez-Nieves, A.; Hu, Z. B.; Weitz, D. A. Angew. Chem., Int. Ed. 2007, 46, 1819–1822. (54) Steinbacher, J. L.; Moy, R. W. Y.; Price, K. E.; Cummings, M. A.; Roychowdhury, C.; Buffy, J. J.; Olbricht, W. L.; Haaf, M.; McQuade, D. T. J. Am. Chem. Soc. 2006, 128, 9442–9447. (55) Liu, K.; Ding, H. J.; Liu, J.; Chen, Y.; Zhao, X. Z. Langmuir 2006, 22, 9453– 9457. (56) Zhang, H.; Tumarkin, E.; Peerani, R.; Nie, Z.; Sullan, R. M. A.; Walker, G. C.; Kumacheva, E. J. Am. Chem. Soc. 2006, 128, 12205–12210. (57) Johann, R. M.; Renaud, P. Biointerphases 2007, 2, 73–79. (58) Rubinstein, M.;Colby, R. H. Polymer Physics; Oxford University Press: New York, 2003. (59) Murthy, V. S.; Rana, R. K.; Wong, M. S. J. Phys. Chem. B 2006, 110, 25619–25627. (60) Rana, R. K.; Murthy, V. S.; Yu, J.; Wong, M. S. Adv. Mater. 2005, 17, 1145–1150. (61) Yu, J.; Murthy, V. S.; Rana, R. K.; Wong, M. S. Chem. Commun. 2006, 10, 1097–1099. (62) Yu, J.; Yaseen, M. A.; Anvari, B.; Wong, M. S. Chem. Mater. 2007, 19, 1277–1284. (63) Kadali, S. B.; Soultanidis, N.; Wong, M. S. Top. Catal. 2008, 49, 251–258.

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Article solution was effective at removing organic materials from the wafers. It is important to note that this solution is very oxidative, corrosive, and possibly explosive and requires careful handling. SU-8 50 photoresist and SU-8 developer solutions were purchased from Microchem Corporation. Poly(dimethylsiloxane) (PDMS) used to make microfluidic channels was prepared from Sylgard 184 kits purchased from Dow Corning. The kit consists of a liquid silicone rubber elastomer base (vinyl-terminated PDMS) and a curing agent (mixture of a platinum complex and copolymers of methylhydrosiloxane and dimethylsiloxane) that were mixed in a weight ratio of 10:1 to result in liquid PDMS. Poly(allylamine hydrochloride) (PAH, Mw = 70 000 g mol-1) was obtained from Fisher Scientific and trisodium salt of citric acid (trisodium citrate or simply citrate) from Sigma-Aldrich. Unless specified otherwise, the pH of PAH and citrate solutions was maintained at the natural pH of preparation. (The pH of PAH was 4.38, and that of trisodium citrate was 8.38). Sodium hydroxide (1 N NaOH) and hydrochloric acid (1 N HCl) used in investigating the dependence of PAH-citric acid interactions on pH were purchased from Fischer Scientific. For fluorescence studies, charged dyes such as fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC) were purchased from Sigma-Aldrich. FITC and RITC dyes were conjugated to PAH for fluorescence studies using a membrane dialysis protocol described elsewhere wherein 1% (w/v) of the PAH was replaced by its conjugated variant.64 Unless otherwise specified, all fluorescence studies utilized PAH conjugated with RITC (called PAH-RITC). A neutral fluorescent dye (dextran-tetramethylrhodamine or dextran-Rh) was procured from Invitrogen Corporation. Studies featuring the neutral dye were also performed at a dye concentration of 1% (w/v). All solutions were prepared using ultrapure-grade water (18.2 MΩ-cm, Barnstead Nanopure Diamond System). 2.2. Microfluidic Device Fabrication. Microfluidic channels used in this study have an architecture that consists of three inlet channels (width  height = 50 μm  50 μm) that merge to form the main channel (width  height = 150 μm  50 μm). The device was fabricated using standard replica molding techniques and photoresist processing guidelines.65-68 First, an inverse layout of the microchannel pattern was generated using conventional photolithography techniques in a class 100 clean room facility (Rice University). Silicon wafers (University Wafers, 4 in. mechanical grade) were precleaned in piranha and dehydrated at 200 °C. A layer of 50-μm-thick negative UV photoresist (SU-8 50) was spun onto the wafer using a spin coater (Headway Research Inc., 2000 rpm for 30 s) and soft baked on a hot plate at 95 °C for 20 min. Next, the device designed using AutoCAD and printed onto a chrome mask (Fineline Imaging) was transferred to the photoresist by a conventional contact aligner (SUSS Mask Aligner MJB4). The photoresist was developed in SU-8 developer solution to remove uncross-linked resist. This gave the master template with positive microchannel relief. In fabricating the microfluidic device, liquid PDMS was poured onto the silicon master, degassed for 20 min, and cured at 80 °C. Holes were punched into the cured PDMS channels, which were then treated with UV/ozone (Novascan) and bound to glass slides. Tubing (SCI Scientific Commodities, 0.58 mm inner diameter) was inserted at the inlet and outlet ports, and the junctions were rendered airtight using epoxy. 2.3. Monitoring Gel Formation. While imaging, the device was placed on the stage of an inverted microscope (Olympus) and reactants flowed at controlled rates using either a syringe pump (64) Yoo, R. M.S. Thesis, Rice University, Houston, TX, 2006. (65) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974–4984. (66) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551–575. (67) Xia, Y. N.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153–184. (68) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H. K.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27–40.

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Article (Harvard Apparatus Pump 33) or a gas displacement pump (Fluigent MFCS 8C), as shown in Figure S1 in Supporting Information. Unless otherwise specified, PAH flowed through the central channel in all studies and citric acid flowed through the side channels. Events were monitored by video microscopy with a digital camera (Hamamatsu) at a rate of 10 frames/s using Simple PCI software. Structures resulting from PAH-citric acid interactions were characterized by optical microscopy (Olympus IX71) and confocal microscopy (Carl Ziess LSM 510). In fluorescence studies, a laser excitation wavelength of 488 nm was chosen for FITC (λEx = 494.5 nm, λEm = 519 nm), and an excitation wavelength of 543 nm was selected for both RITC (λEx=560 nm, λEm =580 nm) and dextran-Rh (λEx =555 nm, λEm =580 nm). Image analysis and intensity profiling of fluorescing structures were carried out using Simple PCI and Image J (National Institutes of Health) software. In studying PAH-citric acid interactions in microchannels, channels were first primed with PAH-RITC drawn through the central channel and ultrapure water drawn through the side channels. After priming, the ultrapure water stream was replaced by citric acid buffer.The initial PAH concentration was 1 mg/mL, and the concentration of citric acid was adjusted so that charge ratio (R) of citric acid to PAH varied from 0.1 to 1 to 10 to 50. (Charge ratio R is defined as the ratio of the total negative charge of the added salt to the total positive charge of the polymer, R = (anion  z-)/(cation  zþ), where z- is the negative charge per molecule of citric acid (3-) and zþ is the positive charge per PAH monomer (1þ). The base polymer concentration for calculations of citric acid at all specified R ratios was 1 mg/mL. The flow of reactants was individually varied at volumetric flow rates of 25 μL/h, 25 μL/min, 25 mL/h, and 25 mL/min (corresponding to flow rates from 0.025 to 1500 mL/h). Although it takes a minimum of 16 experiments to verify the resulting structures (for 4 R ratios and 4 flow rates), most data points were repeated a minimum of 3 times to ensure data reproducibility and to obtain reliable statistics. For cases where the flow rate was 25 μL/h or 25 μL/min, the experiments were repeated twice on account of the lengthy duration of the experiment.

3. Results and Discussion 3.1. Nature of the Structures Formed in Microfluidic Channels. Predominantly, three types of structures (aggregates, gels, and droplets) were seen to form. The coexistence of multiple structures was seen to occur in the microfluidic channel for several of the investigated conditions. Structures observed in the microfluidic device were plotted on a state diagram, shown in Figure 1 as a function of the R ratio and the flow rate of reactants. These nonequilibrium structures correspond to those that emerged within a specific cutoff time interval. Eventually, all conditions led to the formation of a gel phase. The cutoff time was fixed at 5 min, given that most structures formed within this time frame with the exception of a few cases of extreme R ratios or flow rates, where the time required for structure formation spanned across several tens of hours. The cutoff time allowed us to probe the emerging structures and mechanism leading to gel formation. 3.1.a. Citrate-Bridged Aggregates of PAH or PAH-Citrate Aggregates. The first structures that emerged were PAH-citrate aggregates that formed at the interface between the citric acid and PAH streams, as illustrated in Figure 2a-c. These aggregates appeared as micrometer-sized spheres and as bright spherical spots in fluorescence mode, as shown in Figure 2b. Upon crosslinking, the fluorescence intensity of the PAH-citrate aggregates was seen to be significantly higher than the background fluorescence intensity of PAH-RITC. The aggregates that formed were in Brownian motion, which is consistent with earlier studies by Murthy et al., who reported the formation of colloidal aggregates by vortex mixing PAH and citrate under batch conditions.59-63 6652 DOI: 10.1021/la903983y

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Figure 1. Nonequilibrium state diagram summarizing changes in state of PAH from PAH-citrate interactions. (A diagram was constructed from changes observed in the PAH stream within the first 5 min of reacting flow. The initial inlet PAH concentration was fixed at 1 mg/mL in all cases). Regions west and south of the dotted line mark the space where aggregates could be resolved under fluorescence microscopy, and regions north and east of the dashed line mark the space where droplets appeared.

3.1.b. Gel Networks of PAH. After the aggregrates formed, gel networks of PAH cross-linked by citrate appeared as noncontinuous, irregularly shaped nonspherical networks. As with the aggregates, the formation of the noncontinuous gel phase, as shown in Figure 2d-f also began at the interface between the polymer and citrate streams. Eventually, these noncontinuous gel networks formed a continuous gel, as shown in Figure 2g-i. Our microscopy studies also revealed that gelation occurred entirely within the PAH stream, indicating that the gel was very slow in diffusing into the citrate stream. As PAH was cross-linked by citrate, an increase in the fluorescence intensity was observed as the gel phase formed. Also, the viscosity of the PAH gel was significantly higher when compared to that of unreacted PAH and citrate streams. 3.1.c. Droplets of Citrate. Once the gel phase was formed, aqueous droplets rich in citrate ions emerged within the gel, as shown in Figure 2j-l. Most droplets formed at the junction of the PAH and citrate streams as illustrated in Figure 2j,l. A noteworthy feature of droplets that formed was their deformable nature wherein they elongated to ellipsoids under shear flow. Droplets that emerged were found to be unstable in nature, and after a sufficient number density of droplets formed (at high R ratios and high flow rates), they grew to larger droplets and droplet networks as shown in Figure 2k. To establish that droplets were indeed citrate-rich aqueous regions, control experiments on two fluorescent dyes were performed. In the first experiment, PAH conjugated with FITC (PAH-FITC) was flowed through the central stream while a mixture of citrate and neutral dextran-Rh dye was flowed through the side streams, as shown in Figure 3a. PAH conjugated with FITC was selected in this experiment over previously used PAH-RITC because of its appreciable difference in excitation wavelength over that of dextran-Rh. Neutral dextran-Rh was selected to prevent electrostatic interactions of the dye with PAH. Upon droplet formation, the reactant flow was stopped and the channel was imaged under a confocal microscope using FITC and rhodamine filters. Fluorescence imaging revealed that droplets appeared dark under the FITC filter, thus indicating an absence of PAH in the droplets as shown in Figure 3b. Droplets appeared bright under the rhodamine filter, thereby indicating the presence of dextran-Rh inside the droplets, as shown in Figure 3c. Intensity profiling across a magnified region of a representative droplet, as illustrated in Figure 3e,f, was carried out and confirmed the Langmuir 2010, 26(9), 6650–6656

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Figure 2. Optical and fluorescence microscopy images of structures formed in microfluidic channels from PAH-citrate interactions.

(1) PAH-citrate aggregates (formation conditions: R = 10, PAH = 1 mg/mL, flow rate = 25 μL/h): (a) bright field and (b) fluorescence images and (c) schematic of region where aggregates formed in the microfluidic channel (scale bars - a, b: 10 μm). (2) PAH cross-linked incompletely to form noncontinuous gels (formation conditions: R = 0.1, PAH = 1 mg/mL, flow rate = 25 mL/h): (d) bright field and (e) fluorescent images and (f) schematic of the region in the microfluidic channel where discrete gels formed (scale bars - d, e: 10 μm). (3) PAH cross-linked completely to form continuous gels (formation conditions: R = 50, PAH = 1 mg/mL, flow rate = 25 mL/h): (g) bright field and (h) fluorescent images and (i) schematic of the region in the microfluidic channel where continuous gels formed (scale bars - g, h: 50 μm). (4) Droplets rich in citrate ions (formation conditions: R = 50, PAH = 1 mg/mL, flow rate = 25 mL/min): (j) bright field and (k) fluorescent images of citrate droplets within the continuous gel stream of the microfluidic channel. (l) Schematic depicting droplet formation in the microfluidic channel. Droplets emanate from the junction of the microfluidic channel after reactant streams first interact and form continuous gels. The droplets that formed flowed along the length of the channel. (scale bars - j, k: 25 μm).

absence of PAH within the droplet and the presence of dextranRh in regions within the droplet. (The color version of Figure 3 is available as Figure S2 in Supporting Information). A negative control experiment featured the replacement of citrate streams with a water-dextran-Rh stream upon the onset of polymer gelation and droplet formation. It was seen that no droplets emerged, thereby establishing the requirement of citrate for droplet formation (Supporting Information, Figure S3). The citrate solution emerged as droplets only after completely cross-linking the PAH into a continuous gel. A plausible explanation for the emergence of citrate-rich droplets in completely gelated regions of PAH is that the gel remains permeable to the citrate solution. It is noteworthy that the driving force for citrate to diffuse into the PAH stream, even upon completely crosslinking PAH into a continuous gel, is predominantly a concentration gradient, given that the concentration of citrate entering the side streams of the microfluidic channel was fixed. It is possible that the citrate ions exist as a separate phase in citraterich droplets, possibly to screen themselves from the hydrophobic organic groups of cross-linked PAH. The presence of the neutral dextran-Rh dye specifically within the citrate-rich droplet phase and its absence from the gelated PAH region supports the concentration-gradient-driven argument of citrate ion diffusion and the hydrophobic nature of the cross-linked PAH gel that results in the emergence of citrate-rich aqueous droplet phases. 3.2. Structural Changes in Microfluidic Channels. The previous section described the nature of structures from PAHcitrate interactions in microfluidic channels. From the nonequilibrium state diagram, it is evident that multiple structures coexist Langmuir 2010, 26(9), 6650–6656

across all R ratio-flow-rate pairs. Figure 4 is a representative confocal image of the microchannel that clearly illustrates the coexistence of multiple structures. It can be seen from Figure 1 that the following trends emerge for the structural evolution from the PAH-citrate interaction: (1) PAH-citrate aggregates formed under the following conditions: (a) R g 1 when the flow rate was 25 μL/h and (b) R = 0.1 for flow rates G25 μL/min. (2) Polymer gels emerged when (a) R > 1 with a flow rate of 25 μL/h where noncontinuous/continuous gels coexisted with aggregates but not droplets and (b) R = 0.1 with a flow rate G25 μL/min where only noncontinuous gels formed that coexisted with aggregates but not continuous gels or droplets. (3) Droplets emerged for R > 1 when the flow rate was G25 μL/min and coexisted with noncontinuous/ continuous gels. It is noteworthy that as the magnitude of the flow rate increased, the R ratio at which droplets formed decreased (R = 50 for a flow rate of 25 μL/min and R = 10 for a flow rate of 25 mL/h). The following sections explain the effects of charge ratio, flow rate, and pH on structure formation. 3.3. Effects of R Ratio and Flow Rate. The trends shown in Figure 1 indicate that the R ratio and flow rate of reactants affected the time required to form a PAH gel. For a given flow rate, an increase in the R ratio provides a higher concentration of citrate ions to the microfluidic channel. The resulting effect was that aggregates appeared at R = 1 for a flow rate of 25 μL/h, DOI: 10.1021/la903983y

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Figure 3. (a) Schematic to elucidate the nature of droplets. A neutral dye (dextran-tetramethylrhodamine) was added to the citrate stream and reacted with PAH-FITC under conditions of R = 10, PAH = 1 mg/mL, and flow rate = 25 mL/h. Upon droplet formation, the flow was stopped and the reactant streams were observed in fluorescence mode of a confocal microscope. (b) Confocal images of droplets observed under a FITC filter. (c) Confocal images of droplets observed under a rhodamine filter. (d) Differential interference contrast (DIC) image of droplets corresponding to images b and c). (e) Magnified region of a droplet observed through a FITC filter that corresponds to the circled region in image b. (f) Magnified region of a droplet observed through a rhodamine filter that corresponds to the circled region in image c. (g) DIC image of droplets corresponding to images e and f. (h) Intensity profiles of droplets in images e and f along regions marked with a white line establishes the absence of PAH in droplet and the diffusion of dextran dye into the interior of the droplet. (All scale bars are 20 μm).

Figure 4. Schematic depicting the coexistence of structures in a microfluidic channel using confocal microscopy (conditions: R = 10, PAH = 1 mg/mL, and flow rate = 25 mL/h) when viewed under (a) a FITC filter and (b) a rhodamine filter. (c) Predominant existence of PAH-citrate aggregates (green spots) and gel (continuous green region). (d) PAH-citrate aggregates, gels, and citrate droplets (dark spots). (e) Citrate droplets present within the cross-linked polymer gel. (f) Complementary region to image d as viewed through a rhodamine filter. PAH-citrate aggregates appear as dark spots. (g) Complementary region to image e as viewed through a rhodamine filter. The PAH gel appears dark, and droplets of citrate appear red because of the presence of dextran-Rh. (All scale bars are 20 μm).

whereas for a higher R ratio of 50 at the same flow rate, noncontinuous and continuous gels formed along with the aggregates. Given that the flow rates are equal, it is indicative that at higher R ratios there was an increase in the rate of aggregate formation, 6654 DOI: 10.1021/la903983y

which resulted in a faster gelation of PAH. This also explains the general trend of an increase in the emergence of structures observed within the 5 min interval as flow rates were increased for a given R ratio. Langmuir 2010, 26(9), 6650–6656

Kini et al.

An increase in the flow rate for a given R ratio has dual effects: Higher flow rates at a given R ratio increase the concentration of citrate ions in the channel from enhanced convection-mediated transport. Higher flow rates also have an additional effect in that increased shear stresses promote the gelation of PAH from the combination of aggregates. It is important to note that the simple diffusion of citrate in the absence of fluid flow did not promote the gelation of PAH. To elaborate, when the R ratio was fixed at 10, the gelation of PAH occurred when the reactants flowed at 25 mL/min. However, when the flow was halted by equalizing the pressure, no further gelation of PAH was observed downstream from the channel, although the diffusion of citrate ions into the polymer stream continued from the existing concentration gradients and electrostatic interactions. It can also be seen from Figure 1 that in addition to shear stresses from flow rates, a threshold citrate concentration was required to form continuous gels from PAH-citrate aggregates. For the case where the citrate ion concentration was limiting with respect to PAH at R = 0.1, cross-linking of PAH-citrate aggregates to form continuous gels did not occur, even several hours after the onset of reactant flow into the flow channels. This was true across the flow rates (and corresponding shear stresses) investigated ranging from 25 μL/h to 25 mL/min and may be attributed to an inadequate number of aggregates formed when citrate was limiting in concentration, thus rendering incomplete gelation of PAH across the range of flow rates (and shear stresses) investigated (Figure S4 in Supporting Information). 3.4. Effect of Laminar Flow Conditions. A feature of our investigation of polymer gelation in microfluidic channels is that gelation and droplet formation was seen to occur specifically within the PAH stream of the microfluidic device. This may be rationalized from the laminar flow profiles prevalent in the microfluidic environment. It is known that the effective time t for the diffusion of a species i across a length L in a channel is given by ti = L2/Di, where Di is the effective diffusivity of species i (sum of electrostatic and concentration gradient components). It is also known that the diffusivity of a polymer DP (such as PAH, Mw = 70 kDa) is significantly smaller than that of a multivalent ion DC (such as citrate, Mw = 294 Da). Hence, it follows that tP > tC, implying that the time required for citrate ions to diffuse into the PAH stream is much shorter than that required for PAH to diffuse into the stream containing citrate ions. After the citrate ions diffuse into the PAH stream to cross-link the polymer, the viscosity of the resulting polymer gel stream increases and the diffusivity DG of the gel becomes lower than both the diffusivity of unreacted PAH and the diffusivity of unreacted citrate ions (DG